This invention relates generally to Surface Plasmon Resonance (SPR) sensor devices, their use in biological and chemical sensing, and their use in multisensor devices and applications.
Field-based biological and chemical sensors are of increasing importance due to their ability to detect natural and man-made hazards such as environmental contamination, biowarfare agents, explosives, and foodborne pathogens. Optical sensors based on surface plasmon resonance (SPR) are attractive for these applications.
Surface plasmon resonance (SPR) is an optoelectronic phenomenon used to construct sensitive thin-film refractometers which may be readily applied to chemical and biological sensing (Liedberg, B. et al. (1995), xe2x80x9cBiosensing with surface plasmon resonancexe2x80x94how it all started,xe2x80x9d Biosensors and Bloelectron. 10:i-ix; Sambles, J. R. et al. (1991), xe2x80x9cOptical excitation of surface plasmons: an introduction,xe2x80x9d Contemp. Phys. 32:173-183). One common design of an SPR sensor is shown in FIG. 1a. A prism (1) is coated on one side (2) with a 50 nm gold (or silver) film (3). Monochromatic light (4, TM polarized) enters an opposing prism face and strikes the metal-coated face (2) at a range of angles above the critical angle. The reflected light (5) is measured, and reflectivity (intensity of reflected light) as a function of angle is determined. When plotted, this reflectivity spectrum exhibits an attenuation feature centered at a particular angle (FIG. 1b). This angle is sensitive to the refractive index (RI) within approximately one wavelength of the metal-coated surface: If the RI increases, e.g., due to the presence of an analyte, the angle will increase, as illustrated in FIG. 1b for RI=na=1.33-1.38. Measurement of this angle can be used to measure the effective RI of a thin layer adjacent to the metal surface and to detect changes in RI due to changes in type and concentration of analytes present in that layer.
The planar SPR configuration has practical constraints, including awkward sample handling, requirements for index matching and optical inflexibility, that limit its application. For most SPR sensing experiments, the analyte must smoothly and continuously flow over the planar sensing surface. As illustrated in FIG. 1a, a flat flow cell sealed to the metal surface using gaskets and incorporating input and output fittings for tubing connections is typically employed. Because of the complexity of construction of the cell, sample handling can be awkward and leaks and bubble accumulation, which disrupt sensing, are common problems. Because the prism used in the planar configuration can be an expensive glass component, the SPR metal layer is often deposited on a thin disposable glass slide which is index-matched to the prism. The inexpensive slide may then be changed without disturbing the prism. The use of an index-matching fluid introduces additional complexity in the device, difficulty of use, and other problems including potential sensor drift and analyte contamination. The planar SPR configuration is not readily adaptable to multisensing applications (e.g., simultaneous measurement of several sample properties, particularly optical properties). The planar configuration is designed to measure reflectivity only and only one side of the planar sensing surface is optically accessible. This configuration does not allow other types of optical measurements (e.g., transmissivity or fluorescence) which are of interest in multisensing applications.
SPR sensing technology can be adapted for field sensing applications by improving sensor compactness, ruggedness, and ease of use and by improving the optical flexibility of the SPR sensor by facilitating its use for multiple independent optical measurements.
Several improved sensing SPR configurations, including the ultraminiature fiber-optic SPR probe developed by Jorgenson and Yee, S. S. (1993) xe2x80x9cA fiber optic chemical sensor based on surface plasmon resonance,xe2x80x9d Sensors and Actuators B 12:213-220, the SPR lightpipe developed by Karlson et al. (1996), xe2x80x9cFirst-order surface plasmon resonance sensor system based on a planar light pipe,xe2x80x9d Sensors and Actuators B 32:137-141, in which the optical substrate may be replaced without the use of index matching fluid, and a miniature planar probe sensor combining the advantages of these devices (Johnston, K. S. et al. (1999), xe2x80x9cPrototype of a multi-channel planar substrate SPR probe,xe2x80x9d Sensors and Actuators B 54:57-65). Another recent development is a compact, rugged integrated SPR sensor in which all sensor components are contained in one small molded package (Melendez, J. (1997), xe2x80x9cDevelopment of a surface plasmon resonance sensor for commercial applications,xe2x80x9d Sensors and Actuators B 39:375-379).
Several multisensor configurations of SPR devices have been reported. Johnston et al. described how simultaneous measurement of multiple lightpipe sensor xe2x80x9cbandsxe2x80x9d improves the ability of SPR measurements to characterize thin films (Johnston, K. S. (1995), xe2x80x9cNew analytical technique for characterization of thin films using surface plasmon resonance,xe2x80x9d Mater. Chem. Phys. 42:242-246.). Nenninger et al. (1998), xe2x80x9cReference-compensated biosensing using a dual-channel surface plasmon resonance sensor system based on a planar lightpipe configuration,xe2x80x9d Sensors and Actuators B 51:38-45 demonstrated the use of multichannel sensing in the lightpipe geometry to compensate for non-specific binding in SPR biosensing. Chinowsky et al. described the combination of bulk refractive index (RI) measurements with SPR measurements to compensate for interference from temperature or buffer concentration changes (Chinowsky, T. M. and Yee, S. S., U.S. Provisional Application No. 60/132,894, filed May 6, 1999, incorporated by reference herein in its entirety). Chinowsky et al. also demonstrated the use of estimation theory to design optimal linear data analysis techniques for SPR (Chinowsky, T. M. et al. (1999), xe2x80x9cOptimal linear data analysis for surface plasmon resonance biosensors,xe2x80x9d Sensors and Actuators B 54:89-97) and to quantify the ultimate capabilities of such combination measurements (Chinowsky, T. M. and Yee, S. S. (1998), xe2x80x9cQuantifying the information content of surface plasmon resonance reflection spectra,xe2x80x9d Sensors and Actuators B 51:321-330). In related research, Johnston et al. demonstrated a chemometric approach to SPR data analysis and calibration (Johnston, K. S. et al. (1997), xe2x80x9cCalibration of surface plasmon resonance refractometers using locally weighted parametric regression,xe2x80x9d Anal. Chem. 69:1844-1851), and showed that such an approach can enable simplifications in sensor instrumentation (Johnston, K. S. et al. (1999), xe2x80x9cPerformance comparison between high and low resolution spectrophotometers used in a white light surface plasmon resonance sensor,xe2x80x9d Sensors and Actuators B 54:80-88).
The present invention overcomes limitations of currently available SPR configurations by providing a novel device configuration that is less complex, simpler to use and adaptable to multisensor applications.
The present invention provides a capillary SPR sensor. This sensor comprises a capillary substrate, i.e., a tube with an axial cavity, in which at least a portion of the inside surface of the capillary is provided with an SPR-sensing area. The SPR-sensing area comprises an SPR-active conductive layer, which can be a among others, a metal layer (particularly gold or silver), a semiconductor layer or an organic conductor layer. In this SPR sensor configuration, a sample to be analyzed is introduced into the capillary cavity and the capillary substrate is then radially illuminated with light having a TM-polarized component. Light exiting radially from the capillary substrate is detected at selected angles. Radially exiting light that interacts with the SPR-sensing area at angles greater than the critical angle carries SPR features. This light can be detected as a function of incident angle to detect SPR, measure the refractive index of an analyte in the sample, and detect the presence of an analyte in the sample.
In a specific device configuration, an SPR sensor of this invention comprises a substantially transparent capillary substrate having one or more SPR-sensing areas on the inside surface of the capillary, a light source for radially illuminating the SPR-sensing areas at incident angles above the critical angle and a detector for detecting light reflected from one or more SPR-sensing areas of the capillary substrate as a function of incident angle. The light source can be collimated and optionally focused with a lens that is optically coupled to the light source at or near the SPR-sensing area (e.g., the inside surface of the capillary). In operation, the SPR-sensor is provided with means for introducing and removing samples from the capillary substrate. The sensor can be configured for measurement of static samples or for flowing samples.
An SPR-sensing area can comprise a dynamic range-controlling layer adhered to the SPR-active conductive layer to alter the dynamic range of the SPR sensor.
An SPR-sensing area can comprise one or more reactive layers in contact with the SPR-conductive layer which interact with one or more selected analyte species, which may be present in a sample, to change the effective RI detectable by the sensor. A reactive layer can be a biologically reactive layer exhibiting a selective interaction with a biological molecule, e.g., with a selected antigen, protein, peptide, nucleic acid, or related species. A given capillary substrate can be provided with one or more SPR-sensing areas each of which have the same or different reactive layers which are selective for detection of the same or different analytes.
The invention further provides a multisensor comprising a capillary tube substrate having one or more SPR-sensing areas on its inside surface. A multisensor is configured for measurement of at least one optical property of a sample in addition; to SPR. A multisensor of this invention can, for example, be configured to measure bulk RI by critical angle refraction or interferometric methods, fluorescence, chemiluminescence, absorption or Raman scattering of a sample at one or more wavelengths of light. Dependent upon the optical measurements to be performed, the multisensor is provided with one or more light sources to provide for radial or radial and axial illumination of the capillary substrate containing the sample. Also dependent upon the optical measurements to be performed, the multisensor is provided with one or more detectors of radially or axially reflected, refracted, emitted or transmitted light. Dependent again upon the types of optical measurements to be made, the inside surface of the capillary tube substrate is selectively patterned with one or more SPR-sensing areas to provide transparent regions on the inside surface for illumination of the sample in the capillary tube. SPR-sensing areas can be provided at various locations along the inside surface of the capillary and are positioned to allow simultaneous SPR measurement as well as measurement of other optical properties. For simultaneous measurement of SPR and bulk RI only a small region of the capillary need be left uncoated. In specific embodiments, a capillary substrate useful in multisensors of this invention can be provided with an SPR-sensing area on one lengthwise end of the inside surface of the capillary while the other end is uncoated. In an alternate embodiment, a radial section of the inside capillary surface is provided with the SPR-sensing area while the remaining radial section remains uncoated. Similarly, reactive layers can be selectively patterned on the SPR-sensing areas. For example, one or more SPR-sensing areas can be provided with one or more separate reactive layers that are selective for different chemical or biological species.
In specific embodiments, the invention provides multisensor devices which (1) allow measurement of both SPR and bulk refractive index of a given sample; (2) allow measurement of SPR and fluorescence or chemiluminescence of a given sample; or (3) allow measurement of SPR and an absorption or Raman scattering spectrum of a given sample. The invention provides for multisensors in which any one or more emission, refraction, reflection or absorption optical measurements are combined with SPR. Fluorescence or chemiluminescence measurements may require selective labeling of an analyte of interest.
The invention also provides a method for detecting the presence of one or more selected analytes (including biological molecules) in a sample employing an SPR sensor or multisensor device of this invention.
The capillary substrates of this invention can be provided in kits for analysis of selected analytes. A kit contains one or more optionally disposable capillary substrates each provided with one or more SPR-sensing areas which may be patterned on the inside surface of the capillary optionally combined in a kit with analyte control sample(s) for control measurements or device calibration. In specific embodiments, an analysis kit contains one or more capillary substrates provided with one or more SPR-sensing areas each of which sensing areas has a reactive layer selective for a given analyte. A kit can contain one or more capillary substrates having the same or different reactive layers which can be selectively for the same or different analytes. Kits include those that contain capillary substrates having biologically reactive layers that are selective for the same or different biological analytes, e.g., one or more proteins, peptides, nucleic acids or antigens. Kits can also be provided containing one or more capillaries provided with one or more SPR-active conductive layers and reagents for selective introduction of one or more reactive layers on the conductive layers.
The capillary SPR sensor geometry of this invention provides significant improvements in sample handling by providing a capillary sample cell for smooth flow and easy fluid connections; significant improvements in device simplicity and cost by providing one-piece inexpensive optics requiring no index matching and that can employ inexpensive, mass-produced glass tubes as disposable optical sensing elements; and significant improvements in optical flexibility by providing for the use of optical techniques in addition to SPR, such as transmission measurements, bulk refractometry, and fluorescence for analyte analysis.
Multisensor configuration of this invention combine multiple optical measurement technologies in a compact, field-applicable device. These sensors exploit the previously demonstrated benefits of multichannel SPR sensing and bulk RI compensation, while extending the sensor capabilities to include other optical measurement techniques, including bulk and evanescent fluorescence, interferometric RI measurement techniques, absorption, and Raman scattering.
In addition, the use of the capillary substrate protects the SPR-sensing surface from contamination, and (with the addition of end caps) forms a convenient optionally disposable container allowing the environment surrounding the sensing surface to be controlled during storage.
Other advantages and benefits of this invention will be readily apparent on consideration of the following detailed description.